Therapies for treating inflammatory disorders

ABSTRACT

The present invention is directed to an antibody composition for oral administration comprising intact blood-derived polyclonal antibodies that bind to a human tumour necrosis factor α (TNFα), and means for protecting the antibodies during gastrointestinal transit, as well as methods for manufacturing, kits, and therapeutic uses of the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 16/481,439, filed on Jul. 26, 2019, which is the U.S. NationalPhase of on PCT/GB2018/050244, filed on Jan. 29, 2018, which claimspriority to Great Britain Patent Application No. 1701404.4, filed onJan. 27, 2017. The entire content of each of these prior applications isincorporated by reference herein.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 2IX5791_ST25.txt. The text file is 8.90 KB, wascreated on Jul. 19, 2021, and is being submitted electronically viaEFS-Web.

The present invention relates to an antibody therapeutic suitable foruse in treating inflammatory disorders.

Tumour necrosis factor α (TNFα) is a principal cytokine mediatingsystemic inflammation, and is implicated in a number of diseases anddisorders including septic shock, as well as gastrointestinal disorders,such as inflammatory bowel disease.

The two main forms of chronic inflammatory bowel disease (IBD) areulcerative colitis (UC) and Crohn's disease (CD). UC and CD affect morethan five million people in Europe and North America and their incidenceis increasing globally. Both are the result of dysregulation ofintestinal immune homeostasis characterised by markedly elevated levelsof both soluble and membrane-bound TNFα in the intestinal wall. Due tothe chronic nature of these diseases long-term therapy is a requirement.With this in mind, current antibody therapeutics rely on systemicadministration of monoclonal antibodies directed against TNFα. Suchmonoclonal antibodies are typically chimeric or humanised with a view toavoiding induction of a humoral immune response in the patient. The useof polyclonal antibodies of animal origin has been avoided due to therisk of triggering such a response. Three monoclonal antibodiescurrently in widespread use are the chimeric murine Infliximab, andfully humanised Adalimumab and Etanercept. Infliximab is infusedintravenously while the other two are infused intravenously.

Systemic use of antibodies has been associated with infusion reactionsand such treatment is inconvenient for out-patients since intravenousinfusions usually require a short stay in hospital. Additionally, theefficiency with which a systemically administered McAb (Molecular Weight˜150,000 Da) will pass from the blood to the tissue fluid and then crossthe layers of the gastrointestinal tract to reach the inflamed lining ofthe epithelium is also questionable. Moreover, since TNFα is a cytokinethat plays an important pro-inflammatory role systemically in protectingpatients from infection, long-term systemic administration of anti-TNFαantibodies is associated with an increased incidence of seriousside-effects including reactivation of tuberculosis, opportunisticinfections, demyelinating diseases and a long term risk of lymphoma.

The present invention solves at least one of the above-mentionedproblems.

The present inventors have surprisingly found that intact blood-derivedantibodies that bind TNFα, when suitably formulated for oraladministration, constitute an improved therapeutic for inflammatorydisorders, such as IBD.

Polyclonal TNFα antibodies derived from blood, and formulated in acomposition of the present invention when given orally surprisinglyexhibit improved efficacy and exhibit improved specific titres whencompared to conventionally manufactured antibodies (e.g. milk-derivedantibodies).

In particular, polyclonal TNFα antibodies manufactured in an ovine orequine host (preferably ovine host), and formulated in a composition ofthe present invention when given orally, surprisingly exhibit improvedefficacy and improved specific titres when compared to conventionallymanufactured antibodies.

Additionally, or alternatively polyclonal antibodies of the inventionwhen administered orally to a subject elicit no, or greatly-reduced,side-effects (e.g. humoral immune response side-effects) when comparedto conventional antibodies (e.g. monoclonal antibodies) givensystemically. Hence, the composition of the invention is suitable forprolonged therapeutic use, unlike systemically administered antibodycompositions.

As a further advantage, manufacture of said polyclonal antibodies ismuch less expensive than conventional monoclonal antibodies. Hence, thepresent invention provides a scalable and/or cost-efficient therapeutic.

In one aspect the invention provides an antibody composition for oraladministration comprising intact blood-derived polyclonal antibodiesthat bind a human tumour necrosis factor α (TNFα), and means forprotecting the antibodies during gastrointestinal transit.

Advantageously, blood-derived polyclonal antibodies to TNFα can beobtained multiple times from the blood of the same host without killingsaid host. This is in contrast to conventional methods using sourcessuch as bovine colostrum, which only yields antibodies for a limitedtime (e.g. once).

The blood-derived antibodies may be obtainable from a non-human mammal.

The term “obtainable” as used herein also encompasses the term“obtained”. In one embodiment the term “obtainable” means obtained.

Preferably the antibodies are ovine or equine polyclonal antibodies.

More preferably, the antibodies are ovine polyclonal antibodies (e.g.obtainable from an ovine).

Thus, in one aspect there is provided an antibody composition for oraladministration comprising intact ovine polyclonal antibodies that bindto a human tumour necrosis factor α (TNFα), and means for protecting theantibodies during gastrointestinal transit.

An antibody of the present invention binds to and neutralises humantumour necrosis factor α (TNFα). The antibody binds to human TNFα with ahigher binding affinity than for a non-human TNFα or an alternativeantigen. In one embodiment an antibody of the invention binds to humanTNFα with an affinity (measured by the dissociation constant: K_(d)) ofat least 10⁻⁴ M or at least 10⁻⁵ M. In one embodiment an antibody of theinvention may bind to human TNFα with an affinity (K_(d)) of at least10⁻⁶ M or 10⁻⁷ M. Suitably an antibody of the invention may bind tohuman TNFα with an affinity (K_(d)) of at least 10⁻⁸ M or 10⁻⁹ M.

Alternatively or additionally, antibody binding affinity may be measuredby way of the association constant (K_(a)). In one embodiment anantibody of the invention binds to human TNFα with an affinity (measuredby the association constant: K_(a)) of at least 10⁶ M. Suitably anantibody of the invention binds to human TNFα with an affinity (measuredby the association constant: K_(a)) of at least at least 10⁷ M (e.g. atleast 10⁸ M).

Antibody binding can be tested using the assay described in Example 3.Neutralisation can be tested using the assay described in Example 4. Inmore detail, an antibody composition can be assayed to testneutralisation of the cytotoxic effect of TNFα on a L929 mousefibrosarcoma cell line.

Surprisingly, blood-derived antibodies obtainable from a non-humanmammal (preferably an ovine and/or an equine non-human mammal) immunisedwith human TNFα (or a purified fraction thereof) contains a much higherconcentration of antibodies specific for human TNFα, when compared toother non-blood-derived sources (e.g. avian [for example from egg yolk]or bovine sources, such as milk). In some cases, the concentration ofspecific antibodies in said blood (e.g. serum or plasma) or purifiedfraction thereof is 100 times greater.

In one embodiment at least 5% (suitably at least 10%) of the totalantibodies comprised in a blood sample obtainable from a host (e.g.non-human mammal) immunised with human TNFα (or a purified fractionthereof) binds to human TNFα. In another embodiment at least 15% or 20%(suitably at least 25% or at least 30%) of the total antibodiescomprised in a blood sample obtainable from a host (e.g. non-humanmammal) immunised with human TNFα (or a purified fraction thereof) bindsto human TNFα.

In one embodiment at least 5% (suitably at least 10%) of the totalantibodies comprised in antisera obtainable from an ovine immunised withhuman TNFα (or a purified fraction thereof) binds to human TNFα. Inanother embodiment at least 15% or 20% (suitably at least 25% or atleast 30%) of the total antibodies comprised in antisera obtainable froman ovine immunised with human TNFα (or a purified fraction thereof)binds to human TNFα.

In one embodiment at least 5% (suitably at least 10%) of the totalantibodies comprised in blood plasma obtainable from an equine immunisedwith human TNFα (or a purified fraction thereof) binds to human TNFα. Inanother embodiment at least 15% or 20% (suitably at least 25% or atleast 30%) of the total antibodies comprised in blood plasma obtainablefrom an equine immunised with human TNFα (or a purified fractionthereof) binds to human TNFα.

In one embodiment an antibody composition comprises intact polyclonalantibodies at a concentration of 5-100 g/L or 10-75 g/L. In oneembodiment an antibody composition comprises intact polyclonalantibodies at a concentration of 20-75 g/L or 35-60 g/L. Suitably, anantibody composition may comprise at least about 20 or 50 g/L of intactpolyclonal antibodies. The concentrations referred to may be theconcentrations of total intact polyclonal antibodies, suitably totalintact polyclonal IgG (i.e. including antibodies that do, as well as donot, bind to TNFα). In one embodiment the foregoing embodiments refer tointact polyclonal antibodies that bind to TNFα. Preferably theantibodies referred to are ovine polyclonal antibodies.

An “antibody” is a protein including at least one or two, heavy (H)chain variable regions (abbreviated herein as VHC), and at least one ortwo light (L) chain variable regions (abbreviated herein as VLC). TheVHC and VLC regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (FR). The extent of the framework region and CDRs has beenprecisely defined (see, Kabat, E. A., et al. Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242, 1991, and Chothia, C. etal, J. Mol. Biol. 196:901-917, 1987, which are incorporated herein byreference). Preferably, each VHC and VLC is composed of three CDRs andfour FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The VHC or VLC chain of the antibody can further include all or part ofa heavy or light chain constant region. In one embodiment, the antibodyis a tetramer of two heavy immunoglobulin chains and two lightimmunoglobulin chains, wherein the heavy and light immunoglobulin chainsare inter-connected by, e.g., disulphide bonds. The heavy chain constantregion includes three domains, CH1, CH2 and CH3. The light chainconstant region is comprised of one domain, CL. The variable region ofthe heavy and light chains contains a binding domain that interacts withan antigen. The constant regions of the antibodies typically mediate thebinding of the antibody to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system. The term “antibody”includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (aswell as subtypes thereof), wherein the light chains of theimmunoglobulin may be of types kappa or lambda. Preferably an antibodyof the invention is an intact IgG.

The term “intact antibody” is used herein to distinguish an antibody ofthe invention from an antibody fragment (e.g. an antibody Fab, F(ab)₂ orFc). An “intact antibody” therefore comprises (or consists of) each ofthe antibody regions/domains present in a full-length antibody (e.g.obtainable from an ovine). A monomer of an “intact antibody” comprises(or consists of) two heavy chains, and two light chains. The heavychains each comprise (or consist of) a VH domain, a CH1 domain, a CH2domain, and a CH3 domain. The light chains each comprise (or consist of)a CL domain and a VL domain.

Thus, an antibody composition of the present invention comprises no orsubstantially no antibody fragments (e.g. Fab, F(ab)₂ or Fc fragments).The term “substantially no” as used in this context means that less than5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.01% of the total concentration ofantibodies comprised in a composition of the invention are antibodyfragments. Conversely in one embodiment at least 95%, 96%, 97%, 98%,99%, 99.5%, 99.9%, 99.99% or 100% (suitably 100%) of the totalconcentration of antibodies comprised in the composition are intactantibodies. In one embodiment a polyclonal antibody may be purifiedand/or isolated from contaminating antibody fragments.

Advantageously, the intact antibodies of the invention demonstrateimproved TNFα binding and/or neutralisation when compared to antibodyfragments, as demonstrated by an improved TNFα binding capability(Example 3) and/or neutralisation capability (Example 4). Moreover, theintact antibodies of the invention are much less expensive to producethan antibody fragments which require additional processing and/orpurification steps.

In one aspect the invention provides a method for manufacturing intactblood-derived polyclonal antibodies that bind to human tumour necrosisfactor α (TNFα), said method comprising obtaining a blood sample from anon-human mammal that has been administered an immunogen comprisinghuman TNFα or a fragment thereof. The blood sample comprises intactpolyclonal antibodies that bind to human TNFα. The invention alsorelates to antibodies obtainable by said method.

The blood sample may be further processed, e.g. to obtain serum or bloodplasma. The antibody may therefore be obtainable from blood serum orblood plasma. In embodiments where the non-human mammal is an ovine theantibodies may be obtainable from blood serum. In embodiments where thenon-human mammal is an equine the antibodies may be obtainable fromblood plasma.

The term “blood-derived” as used herein means that the antibodies areobtained from the blood of a host (e.g. non-human mammal) used toproduce said antibodies. Typically, blood-derived antibodies may beobtained by administering human TNFα or a fragment thereof to said host(e.g. non-human mammal) subcutaneously, intramuscularly,intraperitoneally, and/or intravenously.

In one embodiment a blood sample (e.g. serum) comprises at least 1, 2,3, 4, 5, 6, 7, or 8 g/L of antibodies that bind to human TNFα,preferably at least 3 or at least 5 g/L of antibodies that bind to humanTNFα.

In one embodiment a blood sample (e.g. serum) comprises about 1 to about12 g/L of antibodies that bind to human TNFα, for example about 3 toabout 9 g/L of antibodies that bind to human TNFα.

The method may further comprise admixing blood-derived polyclonalantibodies with means for protecting said antibodies duringgastrointestinal transit.

In one embodiment a blood sample comprises antibodies that bind to humanTNFα with an avidity of at least 1×10⁹ L/mol, preferably at least 1×10¹⁰L/mol.

Preferably the non-human mammal is an ovine non-human mammal.

Ovine antibodies are antibodies which have been raised in a sheep. Anumber of advantages are associated with using sheep as production hostsfor blood-derived antibodies that that bind to human TNFα. The inventorshave found that the concentration of antibodies that bind to human TNFαpresent in the sheep blood (e.g. serum) remains substantially constantover time, typically taking around 6 months from obtaining a maximumconcentration of specific antibodies for the concentration to halve.Thus, the need for frequent re-immunisation with human TNFα isavoided/minimised. Additionally, the substantially constantconcentration of specific antibodies allows a high yield ofblood-derived antibodies to be obtained per annum. In contrast, theinventors have found that the antibody yield per annum is far lower whenusing milk (e.g. bovine milk), eggs, or colostrum. Moreover, highconcentrations of specific antibodies can be obtained at virtually anytime point in the immunisation schedule (once maximum antibodyconcentrations have been reached). This is in contrast to othernon-human mammals having fluctuating concentrations of specificantibodies in the blood, thus necessitating: measurement of the antibodyconcentration, and ensuring that a blood sample is obtained only whenspecific antibody concentrations are high. Therefore, using sheep asproduction hosts removes this additional step and/or removesunpredictability in the manufacturing method.

In addition, the present inventors have shown that specificblood-derived antibody concentrations between sheep is consistent.

Moreover, sheep are plentiful in the developed world, and easy to workwith when compared to other non-human mammals.

Thus in one embodiment, a non-human mammal is a non-human mammal (e.g.ovine) that has a substantially constant blood concentration ofpolyclonal antibodies that bind to human TNFα after said non-humanmammal has been administered an immunogen comprising human TNFα or afragment thereof.

The term “substantially constant” as used in this context means that theblood concentration of polyclonal antibodies that bind to human TNFαdecreases by 75% or less (preferably by 70%, 65%, or 60% or less, morepreferably by 55% or less (e.g. 50% or less)) of the maximum bloodconcentration of said antibodies (100%) within and/or by 6 months(preferably within and by 6 months) after the maximum bloodconcentration of said antibodies has been reached.

Alternatively or additionally, the term “substantially constant” as usedin this context may mean that within and/or by 6 months (preferablywithin and by 6 months) after the maximum blood concentration ofpolyclonal antibodies that bind to human TNFα has been reached (100%),the blood concentration of said polyclonal antibodies is at least 20% ofthe maximum, preferably at least 25%, 30%, 35%, or 40% or morepreferably at least 45% (e.g. about 50%).

In one embodiment maximum blood concentration of polyclonal antibodiesthat bind to human TNFα occurs at at least 10 weeks from immunisation,such as at least 11 weeks from immunisation. Preferably the maximumblood concentration of polyclonal antibodies that bind to human TNFαoccurs at about 12 weeks from immunisation.

Thus, the present invention includes a method for producing ovineantibodies for use in a composition of the invention, said methodtypically comprising:

-   -   i. administering an immunogen comprising a human TNFα or a        fragment thereof to a sheep;    -   ii. allowing sufficient time for the generation of antibodies in        the sheep; and    -   iii. obtaining the antibodies from the sheep.

The term “sheep” as used herein is synonymous with the term “ovine”. Asused herein, sheep comprise any species that fall within the Ovis genus(e.g. Ovis ammon, Ovis orientalis aries, Ovis orientalis orientalis,Ovis orientalis vignei, Ovis Canadensis, Ovis dalli, Ovis nivicola).

The term “ovine antibody” as used herein is an antibody that has atleast 85%, 90%, 95%, or 99% amino acid sequence identity to an antibodythat has been raised in a sheep. Preferably an “ovine antibody” as usedherein is an antibody that has 100% amino acid sequence identity to anantibody that has been raised in a sheep.

In one embodiment a composition of the present invention comprises onlyovine antibodies, and thus excludes antibodies from a non-ovine source.

The antibody is typically obtainable from the sheep serum. Thus, methodsfor producing ovine antibodies described herein generate sheep antiseracomprising antibodies capable of binding to and/or neutralising humanTNFα. In one embodiment an antibody is isolated and/or purified, forexample isolated and/or purified from a sheep antiserum.

Preferably the non-human mammal is an equine non-human mammal.

Equine antibodies are antibodies which have been raised in a horse.Advantageously, a high yield of blood-derived antibodies that bind tohuman TNFα can be obtained per annum by using horses as productionhosts. Moreover, equine blood cells have been found to settle rapidlyupon collection of a sample, thus avoiding the need for a time-consumingcentrifugation step when obtaining plasma.

In embodiments where the non-human mammal is an equine non-human mammal,the manufacturing method may comprise obtaining blood plasma from ablood sample and returning the blood cells from said sample to saidequine non-human mammal. Suitably, the blood cells may be returned inless than 24 hours, less than 12 hours, less than 6 hours, less than 1hour or less than 30 minutes after obtaining the blood sample.

Thus, the present invention includes a method for producing equineantibodies for use in a composition of the invention, said methodtypically comprising:

-   -   i. administering an immunogen comprising a human TNFα or a        fragment thereof to an equine;    -   ii. allowing sufficient time for the generation of antibodies in        the equine; and    -   iii. obtaining the antibodies from the equine.

The term “horse” as used herein is synonymous with the term “equine”. Asused herein, horses comprise any species that fall within the Equusgenus. Preferably a horse is one or more from the species Equus ferus,such as Equus ferus caballus.

The term “equine antibody” as used herein is an antibody that has atleast 85%, 90%, 95%, or 99% amino acid sequence identity to an antibodythat has been raised in a horse. Preferably an “equine antibody” as usedherein is an antibody that has 100% amino acid sequence identity to anantibody that has been raised in a horse.

In one embodiment a composition of the present invention comprises onlyequine antibodies, and thus excludes antibodies from a non-equinesource.

The antibody is typically obtainable from the horse blood plasma. Thus,methods for producing equine antibodies described herein generate equineblood plasma comprising antibodies capable of binding to and/orneutralising human TNFα. In one embodiment an antibody is isolatedand/or purified, for example isolated and/or purified from an equineblood plasma.

The immunogen used to generate an antibody of the present invention is ahuman TNFα, which has optionally been purified. The term “human TNFα” asused herein encompasses a full-length human TNFα a variant thereof or afragment thereof. Preferably the term “human TNFα” means a full-lengthhuman TNFα. Suitably, the human TNFα may be a recombinant human TNFα.

An immunogen may be a human TNFα comprising (or consisting of) SEQ IDNo. 1. In one embodiment an immunogen is a fragment of SEQ ID No. 1. Inone embodiment an immunogen is a human TNFα variant (or fragmentthereof) having at least 70% (suitably at least 80%) sequence identityto SEQ ID No. 1. Suitably an immunogen is a human TNFα variant (orfragment thereof) having at least 90% (suitably at least 95%) sequenceidentity to SEQ ID No. 1.

An immunogen may be a human TNFα comprising (or consisting of) SEQ IDNo. 2. In one embodiment an immunogen is a fragment of SEQ ID No. 2. Inone embodiment an immunogen is a human TNFα variant (or fragmentthereof) having at least 70% (suitably at least 80%) sequence identityto SEQ ID No. 2. Suitably an immunogen is a human TNFα variant (orfragment thereof) having at least 90% (suitably at least 95%) sequenceidentity to SEQ ID No. 2.

In one embodiment an immunogen comprises (or consists of) SEQ ID No. 1,SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6,SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11,SEQ ID No. 12, or SEQ ID No. 13 or a sequence having at least 70%sequence identity thereto (for example at least 80% sequence identitythereto). In one embodiment an immunogen comprises (or consists of) asequence having at least 90% (e.g. at least 95%) sequence identity toSEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5,SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10,SEQ ID No. 11, SEQ ID No. 12 or SEQ ID No. 13. In one embodiment animmunogen is a fragment or variant of one or more of said sequence(s).

In one embodiment a “variant” may be a mimic of the peptide or peptidefragment, which mimic reproduces at least one epitope of the peptide orpeptide fragment. In another embodiment a “variant” may be a peptide orpeptide fragment having at least one amino acid mutation or modificationwhen compared to a sequence described herein. In one embodiment avariant is SEQ ID No. 13.

The “fragment” referred to herein may be a fragment of SEQ ID No. 1having any number of amino acids from 1 to 156. Alternatively oradditionally the “fragment” referred to herein may be a fragment of SEQID No. 2 having any number of amino acids from 1 to 232. The fragmentpreferably includes at least one epitope of human TNFα. The “fragment”may also have a common antigenic cross-reactivity and/or substantiallythe same in vivo biological activity as the human TNFα from which it isderived. For example, an antibody capable of binding to a fragment wouldalso be capable of binding to the human TNFα from which it is derived.Alternatively, the fragment may share a common ability to induce a“recall response” of a T-lymphocyte which has been previously exposed toan antigenic component of human TNFα.

In one embodiment a fragment comprises (or consists of) SEQ ID No. 1,SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7,SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, or SEQ ID No.12 or a sequence having at least 70% sequence identity thereto (forexample at least 80% sequence identity thereto). In one embodiment afragment comprises (or consists of) a sequence having at least 90% (e.g.at least 95%) sequence identity to SEQ ID No. 1, SEQ ID No. 3, SEQ IDNo. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ IDNo. 9, SEQ ID No. 10, SEQ ID No. 11, or SEQ ID No. 12.

In one embodiment an immunogen or fragment thereof comprises (orconsists of) the N-terminal or N-terminal fragment of human TNFα (e.g.SEQ ID No. 1 or SEQ ID No. 2). In one embodiment the N-terminal orN-terminal fragment comprises (or consist of) SEQ ID No. 3, SEQ ID No.4, SEQ ID No. 5, SEQ ID No. 6 or SEQ ID No. 7 or a sequence having atleast 70% sequence identity thereto (for example at least 80% sequenceidentity thereto). In one embodiment the N-terminal or N-terminalfragment comprises (or consists of) a sequence having at least 90% (e.g.at least 95%) sequence identity to SEQ ID No. 3, SEQ ID No. 4, SEQ IDNo. 5, SEQ ID No. 6 or SEQ ID No. 7.

In some embodiments the polyclonal antibodies of the invention exhibitcross-reactivity to and/or neutralisation of murine TNFα.

Advantageously, antibodies binding to N-terminal fragments of human TNFαmay exhibit improved neutralisation properties.

Without wishing to be bound by theory, it is believed that human TNFαcomprises (or consists of) a plurality of epitopes. For example a humanTNFα monomer may comprise at least 2 or 3 epitopes. Human TNFα is alsobelieved to adopt a trimeric structure. Thus, a human TNFα trimer maycomprise further epitopes. Without wishing to be bound by theory, it isbelieved that at least 5 to 15 antibodies (e.g. 10 to 15 antibodies) ofthe composition may bind to a human TNFα trimer. Suitably about 12antibodies of the composition may bind to a human TNFα trimer.

The antibody composition of the invention comprises polyclonalantibodies, thus preferably said antibody composition comprises apopulation of antibodies wherein the population is capable of binding tomultiple epitopes (preferably all epitopes) of human TNFα.

In one embodiment an antibody composition of the invention comprises afirst antibody that binds to a first epitope of human TNFα and a secondantibody that binds to a second epitope of human TNFα. Preferably, anantibody composition of the invention comprises a third antibody thatbinds to a third epitope of human TNFα. Suitably, an antibodycomposition of the invention may comprise further antibodies, each ofwhich bind to different further epitopes of human TNFα.

An antibody composition of the invention suitably comprises antibodiesthat bind to SEQ ID No. 1 and/or SEQ ID No. 2. Suitably an antibodycomposition of the invention may comprise antibodies that bind to SEQ IDNo. 13.

In one embodiment an antibody composition comprises antibodies that bindto one or more of (e.g. a plurality of) SEQ ID No. 1, SEQ ID No. 2, SEQID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ IDNo. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12 or SEQID No. 13, or a sequence having at least 70% sequence identity thereto(for example at least 80% sequence identity thereto). In one embodimentan antibody composition comprises antibodies that bind to one or more(e.g. a plurality of) sequence(s) having at least 90% (e.g. at least95%) sequence identity to SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ IDNo. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12 or SEQ ID No. 13.

In one embodiment an antibody composition comprises antibodies that bindto the N-terminal of human TNFα (e.g. SEQ ID No. 1 or SEQ ID No. 2). Inone embodiment said antibodies bind to one or more of (e.g. a pluralityof) SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 or SEQ ID No.7 or a sequence having at least 70% sequence identity thereto (forexample at least 80% sequence identity thereto). In one embodiment saidantibodies bind to one or more (e.g. a plurality of) sequence(s) havingat least 90% (e.g. at least 95%) sequence identity to SEQ ID No. 3, SEQID No. 4, SEQ ID No. 5, SEQ ID No. 6 or SEQ ID No. 7.

A plurality of sequences means at least 2 (e.g. at least 3, 4, 5, 6, 7,8, 9, 10, 11 or 12) of said sequences. Suitably the term plurality ofsequences means all of the recited sequences.

Antigens may be formulated with an adjuvant. Suitable adjuvants mayinclude alum (aluminium phosphate or aluminium hydroxide), saponin (andits purified component Quil A), Freund's complete and incompleteadjuvant, RIBBI adjuvant, and other adjuvants used in research andveterinary applications.

The invention contemplates a wide variety of immunisation schedules. Inone embodiment, a non-human mammal (e.g. a sheep or horse) isadministered an immunogen on day zero and subsequently receives animmunogen at intervals thereafter. The interval range and dosage rangerequired can be determined by the person skilled in the art based oninter alia the precise nature of the immunogen, the route ofadministration, and the nature of the formulation. Variations in thesedosage levels can be adjusted using standard empirical optimisationroutines. Similarly, it is not intended that the present invention belimited to any particular schedule for antibody collection. Thecollection time may be typically after day 56. Levels of the specificantibody, i.e. that which binds to the immunogen, may represent at least2 g per litre of blood, serum or plasma (e.g. at least 3 g per litre ofblood, serum or plasma).

The antibodies obtained from the non-human mammal (e.g. sheep or horse)may be subsequently purified thus providing a “purified fraction” asreferred to herein. In one embodiment the antibodies may be purified byprecipitation, chromatography, filtration, or combinations thereof. Thepurification method chosen may be one that enables IgG to remain insolution, co-isolate an inhibitor (e.g. α1-anti-trypsin), co-isolatealbumin, or combinations thereof.

The precipitation may be a sodium sulphate precipitation, or a caprylicacid precipitation. Suitably, the precipitation is a sodium sulphateprecipitation. Advantageously, sodium sulphate precipitation allows forco-isolation of α1-anti-trypsin.

The intact polyclonal antibody of the invention is suitably formulatedwith a buffer. The buffer may include physiological salts such as sodiumcitrate and/or citric acid. A physiological salt may be present in thebuffer at a concentration of 100-200 mM or 125-175 mM. Suitably aphysiological salt may be present in a buffer at a concentration ofapproximately 150 mM (preferably at 153 mM).

The antibody composition of the invention is formulated for oraladministration. Orally administered antibody compositions of theinvention have been found to be associated with one or more of thefollowing unexpected advantages:

-   -   efficacious concentrations of the antibodies reach the desired        area of the gastrointestinal tract more rapidly when        administered orally compared to antibody formulations        administered intravenously and, especially, subcutaneously or        intramuscularly;    -   a large proportion of the polyclonal antibodies reach their        target, therefore allowing for administration of significantly        lower concentrations (e.g. less than 10%), e.g. when compared        with systemic administration where most antibodies remain in the        blood and extracellular fluid compartment of the body;    -   orally administered polyclonal antibodies are believed to be        confined to the lumen and wall of the gastrointestinal tract        with none entering the systemic circulation. This has three        major advantages: i. oral administration does not evoke a        systemic immune response so that administration with        compositions of the invention can be continued indefinitely        without evoking a humoral immune response; ii. for the same        reason there is no risk of acute or delayed hypersensitivity        reactions; and iii. the polyclonal antibodies of the invention        will not neutralise systemically located TNFα, thereby avoiding        the risk of infections such as tuberculosis, and other        complications, including malignancy;    -   oral products receive much less regulatory scrutiny. Indeed, the        consumption of e.g. horse, sheep, or ox plasma as a nutritional        supplement drink provides further testament as to its safety        when orally administered. Furthermore, some oral antibody-based        products are treated as a food;    -   antibodies for oral administration require less purification and        safety testing than those given systemically;    -   manufacture does not require the use of clean room facilities;    -   cost of therapy is reduced significantly for reasons including:        the relatively low manufacturing costs of the polyclonal        antibodies of the invention; the lower concentrations of        antibodies required when administering orally; avoiding the need        for admission to hospital as day cases; and avoiding the need        for nurse/healthcare professional time as is required for        systemic (e.g. IV) administration; and/or    -   oral formulation provides for improved patient convenience,        allowing administration from at the subject's own home and at a        time convenient to him/her. Also, the majority of subjects,        especially the paediatric group, find a pleasantly-flavoured        oral product more acceptable than one administered systemically.

Compositions suitable for oral delivery may be in the form of solutions,suspensions or dry powders which are dissolved or suspended in asuitable vehicle prior to use.

In preparing pharmaceutical formulations, the antibodies can bedissolved in the vehicle, and sterilised for example by filtrationthrough a sterile filter using aseptic techniques before filling intosuitable sterile vials or ampoules and sealing. Advantageously additivessuch as buffering, solubilising, stabilising, preservative orbactericidal or suspending and/or local anaesthetic agents may bedissolved in the vehicle.

Dry powders, which are dissolved or suspended in a suitable vehicleprior to use, may be prepared by filling pre-sterilised ingredients intoa sterile container using aseptic technique in a sterile area.Alternatively the ingredients may be dissolved into suitable containersusing aseptic technique in a sterile area. The containers are sealedaseptically.

In a particularly preferred embodiment, a composition for oraladministration is formulated as a liquid.

A problem with oral administration is ensuring that a sufficientconcentration of functional antibody is transported through thegastrointestinal tract to its target (e.g. the small intestine or thecolon). Factors that may inhibit optimal amounts of functional antibodyreaching the gut include the proteolytic enzymes present in thedigestive secretions, which degrade the antibody molecule. Thus, theantibody composition of the invention comprises means for protecting theantibodies during gastrointestinal transit. Such means counter and/orreduce the undesirable effects that are encountered by the antibodycomposition during transit. Undesirable effects may be attributable tofor example gastrointestinal enzymes (e.g. stomach enzymes such aspepsin) and the chemical environment (e.g. stomach acid).

The term “means for protecting the antibodies during gastrointestinaltransit” as used herein is not intended to encompass aprotective/stabilising amino acid modification (e.g. point mutation,substitution, addition, or deletion) of the antibody polypeptide itselfor a protective/stabilising post-translational modification of theantibody (e.g. glycosylation). However, as an exception, in oneembodiment the means may include a PEGylation moiety covalently attachedto an antibody of the invention, while in an alternative embodimentPEGylation and PEGylation moieties are also excluded. In fact, methodsfor protecting/stabilising an antibody by way of amino acid modificationand post-translational modification are disadvantageous, as theycomplicate the manufacturing process and increase production costs. Forthe avoidance of doubt, the foregoing does not exclude the presence ofprotective/stabilising amino acid modifications of the antibodypolypeptide or protective/stabilising post-translational modificationsof the antibody in addition to the presence of a means for protectingthe antibodies during gastrointestinal transit as described herein.

Preferably the means for protecting the antibodies does not comprise alectin. More preferably, the means for protecting the antibodies is notcomplexed with an antibody of the invention.

There follows a non-limiting description of a variety of embodiments ofsaid means. Each of said embodiments may be employed alone or incombination with each other. Additional means known to a skilled personare encompassed by the present invention, and may also be employed aloneor in combination with any of the following embodiments.

In a particularly preferred embodiment the means for protecting theantibodies described herein comprises at least a protease inhibitor.Advantageously, the present inventors have discovered that a compositioncomprising blood-derived polyclonal antibodies that bind to a humantumour necrosis factor α (TNFα) and a protease inhibitor exhibitsunexpected synergistic effects when treating an inflammatory disorder(i.e. more than the expected additive effects obtained from observationsof treating a subject with an antibody and a protease inhibitor alone).

Without wishing to be bound by theory, it is believed that theepithelial surface of the gastrointestinal tract in subjects withinflammatory disorders loses its normal layer of protective mucus. Thepresent inventors have found that this loss leads to attack of saidepithelial surface by proteolytic enzymes resulting in homeostaticdisruption and/or continued/worsened pathology. The antibody compositionof the present invention surprisingly allows both protection of thegastrointestinal tract from said proteolytic enzymes as well as areduction in inflammation and treatment of other symptoms by way of TNFαbinding.

While (as described below) any protease inhibitors can be used in thepresent invention, in one embodiment the protease inhibitor is asoybean-derived protease inhibitor.

Protease inhibitors selected from the Bowman-Birk inhibitor family ofproteins are particularly preferred. The Bowman-Birk inhibitors areserine protease inhibitors that interact with and inhibit proteases byway of an exposed surface loop, which is typically a disulphide-linkedshort beta-sheet region. Suitable Bowman-Birk inhibitors can be obtainedfrom multiple sources, including legumes (e.g. soybean, lima beans, mungbeans, broad beans, adzuki bean), wheat (e.g. Triticum aestivum), barley(e.g. Hordeum vulgare), rice (e.g. Oryza sativa), nuts (e.g. Arachishypogaea), Coix lachtyma jobi, Setaria italica, Macrotyloma axillaris,Lonchocarpus carpassa, Vicia angustifolia, and Alfalfa. Suitably, aBowman-Birk inhibitor may be obtained from a seed of one or more of saidsources.

In one embodiment a means for protecting the antibodies of thecomposition during gastrointestinal transit comprises a polypeptidewhich binds specifically to and suppresses or inactivates theproteolytic activity of trypsin and/or chymotrypsin. Such means may bean inhibitor of trypsin-1 and/or trypsin-2. Alternatively oradditionally said means may be an inhibitor of chymotrypsin B.

In one embodiment, said inhibitor is a macromolecular inhibitor (e.g. amacromolecular inhibitor having a molecular weight of at least 5 kDa),such as a polypeptide-based inhibitor. By way of example, saidinhibitor(s) may contain a polypeptide loop, which when cleaved byeither trypsin or chymotrypsin causes the inhibitor to bind verystrongly to the protease thus inhibiting the further action of trypsinand/or chymotrypsin.

In one embodiment a means for protecting an antibody duringgastrointestinal transit comprises a means obtainable from an egg, suchas a hen (chicken) egg. More specifically, a means for protecting anantibody during gastrointestinal transit may be an egg white. Suitably,the egg white may be a powdered egg white. Thus, in one embodiment thepresent invention comprises admixing an intact polyclonal antibody withan egg (suitably an egg white, preferably a powdered egg white).

A means obtainable from an egg (e.g. egg white), may be a trypsininhibitor, a chymotrypsin inhibitor or a combination thereof. Thus, themeans may comprise an egg-derived (e.g. egg white-derived) trypsinand/or chymotrypsin inhibitor.

In one embodiment the means present in the egg (e.g. egg white) is oneor more of ovomucoid, ovostatin, ovomacroglobulin, or combinationsthereof. Ovomucoids (Mw 28,500±3,500) are glycoprotein proteaseinhibitors of avian egg white. Said inhibitors have activity when testedagainst bovine trypsin and chymotrypsin. Ovostatins andovomacroglobulins are protease inhibitors found in raw avian egg white.

The means obtainable from an egg may be used at any suitableconcentration. Where the means is a dried egg (e.g. powdered egg white),said dried egg may be present in the composition at a concentration ofat least 25 g/L or at least 35 g/L. In one embodiment the dried egg maybe present in the composition at a concentration of at least 45 g/L orat least 55 g/L. In some embodiments the dried egg may be present in thecomposition at a concentration of between 40 g/L to 80 g/L (suitablybetween 50 g/L to 70 g/L).

In one embodiment a means for protecting an antibody duringgastrointestinal transit comprises α-1-anti-trypsin.

In another embodiment the means is a soybean trypsin inhibitor.

In one embodiment, an inhibitor cocktail may be provided, forconvenience, in the form of colostrum (e.g. bovine). Alternatively (orin addition), the active component(s) thereof may be employed. Colostrumis readily combinable with antibodies to provide a suitable formulationfor oral administration.

In one embodiment, the trypsin inhibitor is a small protein (e.g. Mw5-25 kDa) that is naturally synthesized in the exocrine pancreas whichprevents conversion of trypsinogen to trypsin, so protecting itselfagainst trypsin digestion. Pancreatic trypsin inhibitor competitivelybinds to the active site of trypsin and inactivates it at a very lowconcentration. Examples of trypsin inhibitors suitable for use in thepresent invention include both naturally produced andrecombinantly-produced molecules, such as:

Source Mw Additional information Lima beans 8-10 kDa There are sixdifferent lima bean inhibitors. Bovine 6.5 kDa Kunitz inhibitor is thebest known pancreatic inhibitor. Chymotrypsin is pancreas also inhibitedby this chemical, but less tightly. When extracted from lung tissue,this is known as aprotinin. Ovomucoid ca. 27 kDa Ovomucoids areglycoprotein protease inhibitors found in raw avian egg white. Ovostatinca. 175 kDa Ovostatins (ovomacroglobulins) are protease inhibitors foundin raw avian egg white. Soybeans 20.7-22.3 kDa Soybeans contain severaltrypsin inhibitors. All also bind to and inactivate chymotrypsin.

Natural pancreatic trypsin inhibitors are produced by the acinar cellsand provide security against accidental trypsinogen activation andconsequential unbridled proteolysis. By way of example, theintracellular basic trypsin inhibitor (BPTI) was first crystallized byKunitz and Northrop in 1936. Basic pancreatic trypsin inhibitor (BPTI)forms a very stable 1:1 complex with bovine trypsin between pH 3 and 10,and also human trypsins. Chymotrypsin is also inhibited by BPTI. Soybeantrypsin inhibitor (SBTI) first crystallized by Kunitz (1945) is one ofseveral trypsin inhibitors found in soybeans. The best known preparationis that of Kunitz (Mw 21,500±800; isoelectric point: 4.5). The Kunitzsoybean inhibitor consists of a single polypeptide chain crosslinked bytwo disulfide bridges, and inhibits trypsin mole-for-mole and to alesser extent chymotrypsin. Lima bean trypsin inhibitor (LBI) acts uponboth trypsin and chymotrypsin by forming equimolar complexes. Thetrypsin susceptible binding site is a Lys-Ser peptide bond, whereas thesite of chymotrypsin action is a Leu-Ser bond (Krahn and Stevens 1970).Lima bean trypsin inhibitors (Mw 8,000-10,000) may bechromatographically separated into as many as six variants. All havesimilar but not identical amino acid composition, contain six or sevendisulphide bonds and lack methionine and tryptophan.

By way of further example, Bowman Birk protease inhibitors are a groupof chymotrypsin and trypsin inhibitors produced by Soybeans and a rangeof leguminous plants. They are small disulphide rich proteins of 7-10kDa which are non-toxic to humans and well tolerated. Chymotrypsinpeptide inhibitors which are extremely stable to extremes of pH occur inturtle egg whites. These small peptide inhibitors (approx 13 kDa) formstable complexes with chymotrypsin (Guha et al (1984) J. Bioscience 6:155-163).

In one embodiment, the trypsin and/or chymotrypsin inhibitor(s)component may be an antibody (including a fragment thereof) that bindsto (e.g. specifically binds to) and inactivates the enzymatic activityof trypsin and/or chymotrypsin. Such antibody-based inhibitors may beused as an alternative or in addition to the above non-antibody-basedinhibitors. Thus, an inhibitor combination of an antibody-basedinhibitor and a non-antibody inhibitor may be employed. By way ofexample, a non-antibody inhibitor (e.g. an egg-white derived inhibitor)may be used in combination with an antibody inhibitor where the antibodyinhibits chymotrypsin (and/or trypsin). Similarly, a non-antibodychymotrypsin inhibitor may be used in combination with an antibodyinhibitor where the antibody inhibits trypsin (and/or chymotrypsin).Such antibodies may be prepared routinely.

In one embodiment a trypsin and/or chymotrypsin inhibitor may be presentin the composition at a concentration of at least 25 g/L or at least 35g/L. In one embodiment a trypsin and/or chymotrypsin inhibitor may bepresent in the composition at a concentration of at least 45 g/L or atleast 55 g/L. In some embodiments a trypsin and/or chymotrypsininhibitor may be present in the composition at a concentration ofbetween 40 g/L to 80 g/L (suitably between 50 g/L to 70 g/L).

Alternatively or additionally (preferably additionally) a means forprotecting an antibody during gastrointestinal transit comprises anantacid. In use, said antacid component helps protect the antibodycomponent from the highly acid gastric environment that exists within asubject.

An antacid is any substance, generally a base or basic salt, whichcounteracts stomach acidity. In other words, antacids are stomach acidneutralizers that raise the stomach pH, ideally above pH 5.0, for alimited time period. Antacids perform a neutralization reaction, i.e.they buffer gastric acid, raising the pH to reduce acidity in thestomach.

Examples of suitable antacids for use in the present invention include:aluminum hydroxide (e.g. Amphojel, AlternaGEL); magnesium hydroxide(e.g. Phillips' Milk of Magnesia); aluminum hydroxide with magnesiumhydroxide (e.g. Maalox, Mylanta, Diovol); aluminium carbonate gel (eg.Basaljel); calcium carbonate (e.g. Alcalak, TUMS, Quick-Eze, Rennie,Titralac, Rolaids); sodium bicarbonate (e.g. bicarbonate of soda,Alka-Seltzer); magnesium carbonate; magnesium trisilicate; hydrotalcite(e.g. Mg₆Al₂(CO₃)(OH)₁₆.4(H₂O); Talcid); bismuth subsalicylate (e.g.Pepto-Bismol); alginates (e.g. sodium alginate, alginic acid);magaldrate with simethicone (e.g. Pepsil); any of the above incombination with simethicone for example Asilone, which has three activeingredients, aluminium hydroxide and magnesium oxide neutralise the acidremoving the cause of the pain, and dimethicone.

In one embodiment the antacid is magnesium hydroxide and/or aluminiumhydroxide. The aluminium hydroxide may be added to the composition inthe form of a dried aluminium hydroxide gel. Preferably a composition ofthe invention comprises magnesium hydroxide and aluminium hydroxide.

The antacid may be used at any suitable concentration. In one embodimentan antacid may be present at a concentration of at least 5 g/L or atleast 10 g/L (e.g. per antacid used). In another embodiment an antacidmay be present at a concentration of at least 15 g/L or at least 20 g/L(e.g. per antacid used). In some embodiments between 5 g/L to 40 g/L(suitably between 10 g/L to 30 g/L) of antacid may be used (e.g. perantacid used).

In a preferred embodiment a composition of the invention comprises 5 g/Lto 40 g/L (suitably between 10 g/L to 30 g/L) of magnesium chlorideand/or 5 g/L to 40 g/L (suitably between 10 g/L to 30 g/L) of aluminiumhydroxide.

In one embodiment an antibody composition comprises an antacid moleculeand: a polypeptide which binds specifically to and suppresses orinactivates the proteolytic activity of trypsin and/or chymotrypsin;and/or an antibody that binds to trypsin and/or chymotrypsin andinactivates the protease activity of said trypsin and/or chymotrypsin.

A composition of the invention may comprise one or more antimicrobial.In one embodiment an antimicrobial is methylparaben (E218) and/orpropylparaben (E216). Suitably, a composition of the invention comprisesa combination of methylparaben (E218) and propylparaben (E216).

An antimicrobial (e.g. each antimicrobial) may be present in thecomposition at a concentration of at least 0.2 g/L or at least 0.4 g/L.In one embodiment an antimicrobial is present in the composition at aconcentration of at least 0.6 g/L or at least 1.0 g/L. Suitably, anantimicrobial may be present at a concentration of at least 1.5 g/L orat least 2.0 g/L. In some embodiments the antimicrobial is present at aconcentration between 0.2 g/L to 1.0 g/L. In other embodiments theantimicrobial is present at a concentration between 1.0 g/L to 3.0 g/L(e.g. 1.5 g/L to 2.5 g/L).

A composition of the invention may comprise a suspension stabilityagent, for example glycine. The suspension stability agent (e.g. 200 mMglycine) may be present at a concentration of at least 5 g/L, or atleast 10 g/L. In other embodiments the suspension stability agent (e.g.200 mM glycine) is present at a concentration between 10 g/L to 20 g/L.

Alternatively or additionally, a composition of the invention maycomprise an antifoaming agent, such as simethicone. Said antifoamingagent may be present at a concentration of at least 5 g/L, or at least10 g/L. In one embodiment the antifoaming agent is present at aconcentration of between 5 g/L and 25 g/L (suitably between 10 g/L and20 g/L).

In addition to the above a composition of the invention may comprisesweeteners and/or flavourants, such as vanilla essence, a sugar (e.g.glucose, sucrose, etc.), sugar alcohols, honey, fruit, syrups (e.g.maple syrup, rice syrup, birch syrup, pine syrup, hickory syrup, poplarsyrup, palm syrup, sugar beet syrup, sorghum syrup, corn syrup, canesyrup, golden syrup, barley malt syrup, molasses (treacle), brown ricesyrup, agave syrup, yacon syrup), acesulfame potassium (also known asSunett), alitame (also known as aclame), aspartame (also known as Equalor Nutrasweet), anethole, cyclamate, glycyrrhizin, lo han guo, neotame,perillartine, saccharin (also known as Sweet 'n' Low), stevioside,sucralose (also known as SucraPlus and Splenda), or inulin. In oneembodiment the sweeteners include sodium saccharin and mannitol. In oneembodiment the flavourant includes peppermint oil.

Sweeteners and/or flavourants may be present in a composition of theinvention at a concentration of between 0.1 g/L to 40 g/L, for example0.1 g/L to 30 g/L.

The composition of the invention may comprise a suspending agent, suchas xantham gum. Said suspending agent may be present at a concentrationof at least 1 g/L or 2 g/L. Suitably, said suspending agent may bepresent at a concentration between 1 g/L to 10 g/L, for example 3 g/L to5 g/L.

In one embodiment a composition of the invention may be formulated asper the table below:

Category Material name Concentration Active IgG Intact polyclonal 25-100g/l components antibody (e.g. ovine or equine polyclonal antibody) thatbinds human TNFα Protease inhibitor Egg white dried 40-100 g/l (EWD)Excipients Antacid Magnesium  10-35 g/l hydroxide Aluminium  10-35 g/lhydroxide

Optionally, the composition may further comprise:

Category Material name Concentration Antimicrobial Methylparaben (E218)  0.5-5 g/l Propylparaben (E216) 0.1-1.2 g/l Suspension Glycine (200 mM)   5-25 g/l stability Antifoaming agent Simethicone    5-25 g/lSweeteners Sodium saccharin 0.1-0.8 g/l Mannitol   10-30 g/l FlavourPeppermint oil 0.1-0.5 g/l Suspending agent Xanthan gum    1-10 g/l

In a preferred embodiment, a composition of the invention may beformulated as per the table below:

Category Material name Concentration Active IgG Intact polyclonalantibody   50 g/l components (e.g. ovine or equine polyclonal antibody)that binds human TNFα Protease Egg white dried (EWD)   60 g/l inhibitorExcipients Antacid Magnesium hydroxide 23.4 g/l Aluminium hydroxide 26.4g/l (e.g. as a dried gel)

In a preferred embodiment, a composition of the invention may optionallyfurther comprise:

Category Material name Concentration Antimicrobial Methylparaben (E218)   2 g/l Propylparaben (E216)  0.6 g/l Suspension Glycine (200 mM)   15g/l stability Antifoaming agent Simethicone 16.89 g/l Sweeteners Sodiumsaccharin  0.4 g/l Mannitol   21 g/l Flavour Peppermint oil  0.2 g/lSuspendin agent Xanthan gum    4 g/l

In addition (or alternatively) to the above-described formulationcomponents, the composition may include a physical and/or chemical meansfor protecting the antibody from the acidic environment of the stomachso that an active antibody is ultimately delivered to the intestinalsite of action (e.g. the colon).

By way of example, the antibodies may be encapsulated (e.g. pellets,granular matrices, beads, microspheres, nanoparticles, or liposomes)and/or may be chemically protected (e.g. by PEGylation).

Conventional encapsulation techniques suitable for use in the presentinvention include:

Technique employed Polymer(s) used pH dependent Eudragit L100 and S100Eudragit L100 and S100 Eudragit L100 and S100 Eudragit S, Eudragit FS,Eudragit P4135 F Eudragit L 30 D-55 and Eudragit FS 30 D Time dependentHydroxy propyl methyl cellulose Hydroxyethyl cellulose, ethyl cellulose,microcrystalline cellulose Lactose/ behinic acid Hydroxy propyl methylcellulose Hydroxy propyl methyl cellulose acetate succinate Bacteriadependent/ Chitosan Polysaccharide based Pectin Guar gum Chondroitinsulphate Amylose Alginates

The pH in the terminal ileum and colon (except ascending colon) ishigher than in any other region of the gastrointestinal (GI) tract. Thusa dosage form that disintegrates preferentially at high pH levels isoptimal for site-specific delivery into this region. One of the simplestapproaches for designing a pH-dependent multiparticulate colon-specificdelivery system is enteric coated granules. Enteric coating hastraditionally been used to prevent drug release in the upper GI tract.Enteric coating polymers may be used as both binders and as coatingmaterials for granules. The incorporation of citric acid into thecoating and/or the tablet matrix helps to retard in vitro release and invivo absorption because of the prolongation in disintegration time ofthe core system due to the presence of the acid. Most commonly usedpH-dependent coating polymers for peroral delivery are methacrylic acidcopolymers, Eudragit L100 and Eudragit S100, which dissolve at pH 6.0and 7.0 respectively. The combination of these two polymers in variousratios makes it possible to manipulate drug release within 6.0-7.0 pHrange. Capsules comprising these polymers may be further coated withsolutions of polymethacrylates.

Similarly, excipients such as aqueous hydroxypropyl methyl celluloseacetate succinate as a coating material and citric acid as a pHregulating agent may be added. Glyceryl palmitostearate may be used as aretardant material to formulate controlled release matrices.

Coating formulations (e.g. Eudragit S100) may be further covered with alayer of chitosan HCl. Upon hydration, the capsule shell dissolves andthe chitosan layer forms a gel (internal pH of 4.5), which generates anacidic environment around the Eudragit film so that it does not dissolvein the ascending colon. In the ascending colon, the chitosan HCl gel isdegraded by the colonic micro flora, thereby exposing the Eudragit filmto the colonic environment. But since the ascending colon is weaklyacidic with a pH is less than 7.0, the film coat still remains intact.However, on arrival in the descending colon where pH is greater than 7,the Eudragit film coat dissolves and the drug is released in acontrolled fashion from the matrices. Multi-layer coats may be employedbased on, for example, an inner coat (a combination of Eudragit RL/RS),and an outer coat (Eudragit FS 30D). Eudragit FS 30D is an-ionicco-polymer of methyl acrylate, methyl methacrylate and methacrylic acidand is pH sensitive and dissolves at pH above 6.5.

Microbially-controlled delivery systems may also be employed, which relyon the unique enzymatic ability of the colonic micro flora. Deliverysystems of this type enable a more specific targeting, independent of pHvariations along the GI tract. Many natural polysaccharides such aschondroitin sulphate, pectin, dextran, guar gum etc. may be employed.Multiparticulate systems comprising hydrogel beads (chitosan andtripolyphosphate (TPP)) are one option—TPP acts as a counter ion topositively charged chitosan to form gel beads. The beads are loaded withbovine serum albumin (BSA), a protein that is liable to degradation inthe upper parts of GI tract, and the cross-linking of chitosan with TPPresults in reduced solubility of chitosan, thereby resulting in lesserprotein (antibody) release during upper GI transit. Amylose is aparticularly good film-forming polymer (via gelation), and may also bemixed with Eudragit RS/RL 30D aqueous dispersions. Similarly, amidatedlow methoxy pectin which forms rigid gels with divalent cations (e.g.calcium or zinc) may be employed to produce calcium pectinate gel beadsfor colonic delivery. Pectin may be combined with calcium salts—calciumpectinate (the insoluble salt of pectin) is not degraded by gastric orintestinal enzymes but is capable of degradation by colonic pectinolyticenzymes. As an alternative to crosslinking of soluble polysaccharides toform insoluble salts, the polysaccharide based system may be coated withpH sensitive polymers. By way of example, chitosan microcores may beprepared and coated with acrylic polymers, such as Eudragit L100 andEudragit S100 respectively. Eudragit P-4135 F represents a furtherexample of a suitable pH-sensitive polymer, which may be employed toprepare microparticles for colonic delivery.

Multiparticulate systems may be employed, which combine pH sensitivedelivery and biodegradation in the colonic environment. By way ofexample, an inner entrapment matrix of chitosan microcores may beprepared using a technique such as spray drying, followed by applicationof chitosan microcores microencapsulated within Eudragit polymers by atechnique such as oil-in oil solvent evaporation. Upon dissolution ofthe outer Eudragit coat at appropriate pH the exposed chitosanmicrocores swell and form a gel barrier in alkaline pH, and, in thecolonic region, the chitosan undergoes degradation thereby enhancingrelease. Similar colonic delivery multiparticulate systems may be basedon chitosan microspheres coated with Eudragit L100 or S100. Suitablepreparation techniques include emulsion solvent evaporation. Thechitosan may be cross-linked with glutaraldehyde.

Polyacrylates represent a further example of a suitable delivery vehiclefor use in the present invention. By way of example, a terpolymer ofstyrene and hydroxyethyl methacrylate cross-linked with a difunctionalazo-compound may be employed. The system depends on cleavage of the azobond by colonic microflora resulting in degradation of polymer.Similarly, a pH responsive poly (methacrylic-g-ethylene glycol) hydrogelmay be employed as an oral delivery vehicle. Once inside the basic andneutral environment of the small intestine, the gels rapidly swell anddissociate.

In another embodiment, a microcapsule formulation may be employed forperoral colon-specific delivery. In more detail, aqueous colloidalterpolymers of ethylacrylate/methyl methacrylate/2-hydroxyl ethylmethacrylate (poly (EA/MME/HEMA), for example as synthesized by emulsionpolymerization technique(s) may be employed. These polymers exhibitdelayed release profiles which were characterized by a long lag time andsubsequent rapid release of the entrapped moiety.

In another embodiment, orally administered nanoparticles may serve assuitable delivery vehicles. By way of example, loaded nanoparticles maybe entrapped into pH sensitive microspheres, which serve to deliver theincorporated nanoparticle to the desired colonic site of action.Nanoparticles have a large specific surface, which is indicative of highinteractive potential with biological surfaces. Thus, bioadhesion can beinduced by binding nanoparticles with different molecules. By way ofexample, nanoparticles may be prepared from gliadin protein isolate fromwheat gluten and then conjugated with lectins (glycoproteins ofnon-immune origin which provide specific bioadhesion). Accordingly,nanoparticles are provided, which have a high capacity for non-specificinteraction with intestine and the binding of lectin provided greaterspecificity for colonic mucosa.

In one embodiment, a delivery vehicle based on an albumin-chitosan mixedmatrix microsphere-filled coated capsule formulation may be employed. Inthis regard, an antibody preparation of the invention is filled intohard gelatin capsules and enteric coated.

In one embodiment, albumin microspheres may be employed as the oraldelivery system.

In one embodiment, squalane oil-containing multiple emulsions may beemployed

In one embodiment, poly(lactide-co-glycolide) microspheres may beemployed as the oral delivery vehicle.

In one embodiment, a colonic delivery coating comprising a mixture ofpH-responsive enteric polymer (Eudragit S) and biodegradablepolysaccharide (resistant starch) in a single layer matrix film may beemployed. Examples of these delivery vehicles are availablecommercially, such as from Encap Drug Delivery (Livingston,UK)—particular embodiments include PHLORAL™ and ENCODE™.

In addition (or alternatively) to the above delivery vehicle embodiment,the antibodies of the present invention may be protected from aciderosion by PEGylation with polyethylene glycol (PEG). PEG of variousmolecular weights (500-40000Da) may be coupled to IgG, for example, in aratio of 2-20 PEG molecules per antibody molecule. We refer toGreenwald, R. B et al (2003) “Effective drug delivery by PEGylated drugconjugates”, Advanced Drug Delivery Reviews 55, pp.217-250. Thispublication is incorporated in its entirety by reference thereto.

In one embodiment, delivery capsules such as liposomes, micro- ornanocapsules (e.g. chitosan nanocapsules) may be chemically modifiedwith poly(ethylene glycol) (PEG). The typical degree of PEGylation is inthe range of 0.1% to 5%, such as 0.5% to 2%, for example 0.5% or 1%. Thepresence of PEG, whether alone or grafted to chitosan, improves thestability of the delivery capsules in the gastrointestinal fluids.

In one embodiment, the antibodies of the present invention may betreated with monomethoxypoly(ethylene) glycols activated by cyanuricchloride, succinimidyl succinate, and tresyl chloride.

PEGylated delivery vehicles such as liposomes, micro- or nanocapsuleshave an intrinsic ability to accumulate at disease sites and facilitatetransfection of target cells. Unlike many viral vectors, PEGylatedliposomes are generally considered to be non-immunogenic.

In one embodiment, a branched PEGylating reagent is employed as branchedPEG protecting groups are more effective than linear PEG molecules.

In one aspect the invention provides an antibody composition of theinvention for use in treating an inflammatory disorder, wherein theantibody composition is orally administered to a subject. There is alsoprovided a related use of the antibody composition in the manufacture ofa medicament for treating an inflammatory disorder, as well as methodsof treating an inflammatory disorder comprising orally administering anantibody composition of the invention to a subject.

The term “disorder” as used herein also encompasses a “disease”. In oneembodiment the disorder is a disease.

The term “subject” as used herein refers to a mammal, such as a human orother animal. Preferably “subject” means a human subject.

A means for protecting an antibody during gastrointestinal transit maybe orally administered sequentially (e.g. as separate components) orsimultaneously with an antibody of the invention. When theadministration is sequential, preferably the means for protecting isadministered prior to an antibody of the invention. In a preferredembodiment a means for protecting is administered simultaneously with anantibody of the invention.

An antibody composition according to the present invention may be usedto treat an inflammatory disorder selected from: septic shock, agastrointestinal disorder (such as an intestinal disorder), aninflammatory bowel disease, a graft vs. host disorder, and/or aninflammatory disorder caused/exacerbated by: intestinal infection,non-steroidal anti-inflammatory drugs, stress, alcohol, bowel surgery,ischaemia and reperfusion, food allergy, or combinations thereof.

In a preferred embodiment the inflammatory disorder is an inflammatorybowel disease selected from ulcerative colitis, Crohn's disease, or acombination thereof

In a particularly preferred embodiment the inflammatory disorder isulcerative colitis and the antibody is an ovine or equine polyclonalantibody (more preferably an ovine polyclonal antibody).

Inflammatory bowel disease is typically associated with symptoms such asfrequent diarrhoea. Owing to reduced retention of a therapeutic in theintestine, high concentrations of antibody are required for treatment ofinflammatory bowel disease. Monoclonal antibody therapy is thereforenon-viable (e.g. due to high costs associated with production ofmonoclonal antibodies). Advantageously, a polyclonal antibodycomposition of the present invention provides a solution to the problemof treating inflammatory bowel diseases. Said polyclonal antibodycomposition is comparatively inexpensive to manufacture, thus providinga viable therapeutic.

A suitable therapeutic is required to exhibit high specificity for humanTNFα. Advantageously, the blood-derived polyclonal antibodies (e.g.ovine or equine) (owing to the method of manufacture) demonstrate highspecificity for human TNFα when compared to non-blood derived (and/ore.g. non-ovine or non-equine) antibodies and/or monoclonal antibodies.Furthermore, by definition, a composition comprising monoclonalantibodies will only bind to a single epitope whereas a compositioncomprising polyclonal antibodies will bind to several, therebyincreasing the efficacy with which the human TNFα is neutralised.

The term “treat” or “treating” as used herein encompasses prophylactictreatment (e.g. to prevent onset of a disease) as well as correctivetreatment (treatment of a subject already suffering from a disease).Preferably “treat” or “treating” as used herein means correctivetreatment.

The term “treat” or “treating” as used herein refers to the disorderand/or a symptom thereof.

Therefore a composition of the invention may be administered to asubject in a therapeutically effective amount or a prophylacticallyeffective amount.

A “therapeutically effective amount” is any amount of the antibody,which when administered alone or in combination to a subject fortreating an inflammatory disorder (or a symptom thereof) is sufficientto effect such treatment of the disorder, or symptom thereof.

A “prophylactically effective amount” is any amount of the antibodythat, when administered alone or in combination to a subject inhibits ordelays the onset or reoccurrence of an inflammatory disorder (or asymptom thereof). In some embodiments, the prophylactically effectiveamount prevents the onset or reoccurrence of an inflammatory disorderentirely. “Inhibiting” the onset means either lessening the likelihoodof an inflammatory disorder's onset (or symptom thereof), or preventingthe onset entirely.

An appropriate dosage range is one that produces the desired therapeuticeffect (e.g. wherein the antibody/composition is dosed in atherapeutically or prophylactically effective amount). A typical dosageregimen may comprise administering a composition of the invention once,twice, three times, four times, five times, six times or seven times perweek. In one embodiment a dosage regimen comprises administering acomposition of the invention daily, for example once or twice daily.

In one embodiment a composition of the invention is administered to asubject twice daily for the first four weeks of treatment. In someembodiments subsequent treatment is by way of once daily administrationof the composition.

An appropriate dose may be 2 g or less (e.g. 1 g or less) of polyclonalantibodies. In one embodiment a dose is 0.5 g or less or 0.25 g or lessof polyclonal antibodies.

In one embodiment intact polyclonal antibodies are administered at adose of 2 g or less daily. In another embodiment the dose is 1 g or lessdaily, for example 0.5 g or 0.25 g or less daily.

Suitably, the doses above refer to the total amount of intact polyclonalantibodies comprised in a composition of the invention, suitably totalintact polyclonal IgG (i.e. including antibodies that bind to TNFα andantibodies that do not bind to TNFα). The intact polyclonal antibodiesmay be obtainable directly from a blood sample (e.g. antisera) or apurified fraction thereof. Preferably, the intact polyclonal antibodieshave been purified from a blood sample (e.g. an ovine blood sample),such as antisera. For example, the sample may have been subjected toprecipitation (such as sodium sulphate precipitation), and filtration.Suitably, at least 5% (e.g. at least 10%) of the total intact polyclonalantibodies comprised in the composition bind to TNFα. More preferably atleast 15% or at least 20% (such as at least 25% or at least 30%) of thetotal intact polyclonal antibodies comprised in the composition bind toTNFα.

It is surprising that a therapeutic/prophylactic effect is observed atsuch low doses of total intact blood-derived polyclonal antibody, and isdemonstrative of the high concentration of specific antibody (i.e. thatbinds to TNFα) produced when compared to non-blood-derived source, suchas milk. It is furthermore surprising that a therapeutic/prophylacticeffect is observed at such low doses of total intact ovine or equine(preferably ovine) polyclonal antibody, and is demonstrative of the highconcentration of specific antibody (i.e. that binds to TNFα) producedusing an ovine or equine (preferably ovine) host.

In one embodiment, typical daily dosages are in the range of 5-20 mg(e.g. 8-15 mg or approximately 10 mg) of intact polyclonal antibodiesper kg of body weight. The unit dosage can vary from less than 100 mg,but typically will be in the region of 250-500 mg per dose. Said dosemay be administered daily (e.g. 1×, 2×, 3× or 4× per day).

In some embodiments an antibody composition of the invention may beadministered to a subject in combination with one or more furthertherapeutic(s). Said one or more further therapeutic(s) may beadministered sequentially or simultaneously with an antibody compositionof the invention.

In one embodiment an antibody composition is administered in combinationwith a therapeutic that treats an inflammatory disorder (e.g. aninflammatory disorder referred to herein). Suitably, an antibodycomposition may be administered in combination with a therapeutic thattreats an inflammatory bowel disorder (e.g. inflammatory bowel disease).

In one embodiment a therapeutic may be an aminosalicylate (5-ASA), acorticosteroid, an immunomodulator, an antibiotic or a biologicaltherapy (e.g. a therapeutic antibody).

Examples of suitable therapeutics may include: prednisone, prednisolonesodium phosphate, budesonide, mesalamine, sulfasalazine, corticotropin,azathioprine, infliximab, hydrocortisone, methylprednisolone,methylprednisolone sodium succinate, mercaptopurine, dexamethasone,dexamethasone sodium phosphate, betamethasone acetate, betamethasonesodium phosphate, cyclosporine, cromolyn, mycophenolate mofetil,hydrocortisone sodium succinate, hydrocortisone acetate, triamcinoloneacetonide, cortisone, methylprednisolone acetate, or combinationsthereof, or pharmaceutically-acceptable salts thereof.

In one embodiment an antibody composition may be administered with oneor more probiotic(s). The term “probiotic” as used herein means any livemicro-organism(s) (including bacteria or yeasts for example) which, whenfor example ingested or locally applied in sufficient numbers,beneficially affects the host organism, i.e. by conferring one or moredemonstrable health benefits on the host organism. Probiotics mayimprove the microbial balance in one or more mucosal surfaces. Forexample, the mucosal surface may be the gastrointestinal tract (e.g. theintestine).

The invention also provides a kit comprising an antibody composition ofthe invention, and instructions for use of same. The instructions may befor the use of the antibody composition in medicine, suitably for theuse of the antibody composition in treating an inflammatory disorder. Inone embodiment the instructions describe oral administration of theantibody composition to a subject. The instructions may alternatively oradditionally describe an appropriate dosage regimen, for example anydosage regimen described herein.

In one embodiment the kit comprises a first container comprising theantibodies of the invention, a second container comprising means forprotecting said antibodies during gastrointestinal transit, andinstructions. Preferably the instructions describe a method forformulating the antibodies and means to obtain a composition of theinvention.

In one embodiment a kit comprises one or more further therapeutic(s)described herein.

In another aspect the invention relates to a foodstuff comprising anantibody composition of the invention. The foodstuff may be anyfoodstuff in which the antibodies comprised in the antibody compositionremain functional or in which a substantial concentration of saidantibodies retain the ability to bind to and neutralize human TNFα. Theterm “substantial concentration” as used in this context means that atleast 90%, for example at least 95% or 98% of the starting antibodiesretain the ability to bind to and neutralize human TNFα.

The foodstuff may be a dairy product. In one embodiment a dairy productis selected from yogurt, cheese, or milk.

The foodstuff may comprise a probiotic, and optionally one or moreprebiotic. The term “prebiotic” as used herein refers to anon-digestible food ingredient that beneficially affects the host byselectively stimulating the growth and/or the activity of one or morebeneficial bacteria.

Sequence Comparison

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences may be compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequent coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percentage sequence identityfor the test sequence(s) relative to the reference sequence, based onthe designated program parameters.

Optimal alignment of sequences for comparison may be conducted, forexample, by the local homology alignment algorithm of Smith and Waterman[Adv. Appl. Math. 2: 484 (1981)], by the algorithm of Needleman & Wunsch[J. Mol. Biol. 48: 443 (1970)] by the search for similarity method ofPearson & Lipman [Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988)], bycomputer implementations of these algorithms (GAP, BESTFIT, FASTA, andTFASTA—Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705), or by visual inspection [see CurrentProtocols in Molecular Biology, F. M. Ausbel et al, eds, CurrentProtocols, a joint venture between Greene Publishing Associates, In. AndJohn Wiley & Sons, Inc. (1995 Supplement) Ausbubel].

Examples of algorithms suitable for determining percent sequencesimilarity are the BLAST and BLAST 2.0 algorithms [see Altschul (1990)J. Mol. Biol. 215: pp. 403-410; and “http://www.ncbi.nlm.nih.gov/” ofthe National Center for Biotechnology Information].

In one homology comparison, the identity exists over a region of thesequences that is at least 10 or 20 or 30 or 40 or 50 amino acidresidues in length. In another homology comparison, the identity existsover a region of the sequences that is at least 60 or 70 or 80 or 90 or100 amino acid residues in length.

Any of a variety of sequence alignment methods can be used to determinepercent identity, including, without limitation, global methods, localmethods and hybrid methods, such as, e.g., segment approach methods.Protocols to determine percent identity are routine procedures withinthe scope of one skilled in the art. Global methods align sequences fromthe beginning to the end of the molecule and determine the bestalignment by adding up scores of individual residue pairs and byimposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W,see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving theSensitivity of Progressive Multiple Sequence Alignment Through SequenceWeighting, Position-Specific Gap Penalties and Weight Matrix Choice,22(22) Nucleic Acids Research 4673-4680 (1994); and iterativerefinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracyof Multiple Protein. Sequence Alignments by Iterative Refinement asAssessed by Reference to Structural Alignments, 264(4) J. Mol. Biol.823-838 (1996). Local methods align sequences by identifying one or moreconserved motifs shared by all of the input sequences. Non-limitingmethods include, e.g., Match-box, see, e.g., Eric Depiereux and ErnestFeytmans, Match-Box: A Fundamentally New Algorithm for the SimultaneousAlignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992);Gibbs sampling, see, e.g., C. E. Lawrence et al., Detecting SubtleSequence Signals: A Gibbs Sampling Strategy for Multiple Alignment,262(5131) Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle etal., Align-M—A New Algorithm for Multiple Alignment of Highly DivergentSequences, 20(9) Bioinformatics: 1428-1435 (2004).

Thus, percent sequence identity is determined by conventional methods.See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992.Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “blosum 62” scoring matrix of Henikoff and Henikoff (ibid.) asshown below (amino acids are indicated by the standard one-lettercodes).

A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2 −2 1 6C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3 −2 −2 6H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2 −3 −4 −1−2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3 −1 0 −2−3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2 −2 −1 −3−1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1 −2 −1 4 T0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4 −2 −2 −3 −2−2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1 −1 −2 −1 3−3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0 −3 −1 4

The percent identity is then calculated as:

$\frac{{Total}\mspace{14mu}{number}\mspace{14mu}{of}\mspace{14mu}{identical}\mspace{14mu}{matches}}{\begin{bmatrix}\begin{matrix}{{length}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{longer}\mspace{14mu}{sequence}\mspace{14mu}{plus}\mspace{14mu}{the}} \\{{number}\mspace{14mu}{of}\mspace{14mu}{gaps}\mspace{14mu}{introduced}\mspace{14mu}{into}\mspace{14mu}{the}\mspace{14mu}{longer}}\end{matrix} \\{{sequence}\mspace{14mu}{in}\mspace{14mu}{order}\mspace{14mu}{to}\mspace{14mu}{align}\mspace{14mu}{the}\mspace{14mu}{two}\mspace{14mu}{sequences}}\end{bmatrix}} \times 100$

Substantially homologous polypeptides are characterized as having one ormore amino acid substitutions, deletions or additions. These changes arepreferably of a minor nature, that is conservative amino acidsubstitutions (see below) and other substitutions that do notsignificantly affect the folding or activity of the polypeptide; smalldeletions, typically of one to about 30 amino acids; and small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue, a small linker peptide of up to about 20-25 residues, or anaffinity tag.

Conservative amino acid substitutions may include:

Basic: arginine

-   -   lysine    -   histidine Acidic: glutamic acid    -   aspartic acid

Polar: glutamine

-   -   asparagine

Hydrophobic: leucine

-   -   isoleucine    -   valine

Aromatic: phenylalanine

-   -   tryptophan    -   tyrosine

Small: glycine

-   -   alanine    -   serine    -   threonine    -   methionine

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline and α-methyl serine) may be substituted for amino acidresidues of the polypeptides of the present invention. A limited numberof non-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted forpolypeptide amino acid residues. The polypeptides of the presentinvention can also comprise non-naturally occurring amino acid residues.

Non-naturally occurring amino acids include, without limitation,trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline,trans-4-hydroxy-proline, N-methylglycine, allo-threonine,methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine,nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline,2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and4-fluorophenylalanine. Several methods are known in the art forincorporating non-naturally occurring amino acid residues into proteins.For example, an in vitro system can be employed wherein nonsensemutations are suppressed using chemically aminoacylated suppressortRNAs. Methods for synthesizing amino acids and aminoacylating tRNA areknown in the art. Transcription and translation of plasmids containingnonsense mutations is carried out in a cell free system comprising an E.coli S30 extract and commercially available enzymes and other reagents.Proteins are purified by chromatography. See, for example, Robertson etal., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol.202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al.,Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method,translation is carried out in Xenopus oocytes by microinjection ofmutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti etal., J. Biol. Chem. 271:19991-8, 1996). Within a third method, E. colicells are cultured in the absence of a natural amino acid that is to bereplaced (e.g., phenylalanine) and in the presence of the desirednon-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). Thenon-naturally occurring amino acid is incorporated into the polypeptidein place of its natural counterpart. See, Koide et al., Biochem.33:7470-6, 1994. Naturally occurring amino acid residues can beconverted to non-naturally occurring species by in vitro chemicalmodification. Chemical modification can be combined with site-directedmutagenesis to further expand the range of substitutions (Wynn andRichards, Protein Sci. 2:395-403, 1993).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for amino acid residues ofpolypeptides of the present invention.

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244: 1081-5, 1989). Sites of biological interactioncan also be determined by physical analysis of structure, as determinedby such techniques as nuclear magnetic resonance, crystallography,electron diffraction or photoaffinity labeling, in conjunction withmutation of putative contact site amino acids. See, for example, de Voset al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol.224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. Theidentities of essential amino acids can also be inferred from analysisof homologies with related components (e.g. the translocation orprotease components) of the polypeptides of the present invention.

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner etal., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Neret al., DNA 7:127, 1988).

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner etal., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Neret al., DNA 7:127, 1988).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, NewYork (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, NY (1991) provide the skilled person with ageneral dictionary of many of the terms used in this disclosure.

This disclosure is not limited by the exemplary methods and materialsdisclosed herein, and any methods and materials similar or equivalent tothose described herein can be used in the practice or testing ofembodiments of this disclosure. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, any nucleic acidsequences are written left to right in 5′ to 3′ orientation; amino acidsequences are written left to right in amino to carboxy orientation,respectively. The headings provided herein are not limitations of thevarious aspects or embodiments of this disclosure. Amino acids arereferred to herein using the name of the amino acid, the three letterabbreviation or the single letter abbreviation. The term “protein”, asused herein, includes proteins, polypeptides, and peptides. As usedherein, the term “amino acid sequence” is synonymous with the term“polypeptide” and/or the term “protein”. In some instances, the term“amino acid sequence” is synonymous with the term “peptide”. In someinstances, the term “amino acid sequence” is synonymous with the term“enzyme”. The terms “protein” and “polypeptide” are used interchangeablyherein. In the present disclosure and claims, the conventionalone-letter and three-letter codes for amino acid residues may be used.The 3-letter code for amino acids as defined in conformity with theIUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is alsounderstood that a polypeptide may be coded for by more than onenucleotide sequence due to the degeneracy of the genetic code.

Other definitions of terms may appear throughout the specification. Itis to be understood that this disclosure is not limited to particularembodiments described, and as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present disclosure will be defined only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin this disclosure. The upper and lower limits of these smallerranges may independently be included or excluded in the range, and eachrange where either, neither or both limits are included in the smallerranges is also encompassed within this disclosure, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in this disclosure. It must benoted that as used herein and in the appended claims, the singular forms“a”, “an”, and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “an antibody”includes a plurality of such candidate agents and reference to “theantibody” includes reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth. Thepublications discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that such publications constitute priorart to the claims appended hereto.

The invention will now be described, by way of example only, withreference to the following Figures and Examples.

FIGURES

Embodiments of the invention will now be described, by way of exampleonly, with reference to the accompanying Figures, in which:

FIG. 1 shows inhibition of bovine trypsin by Intact Anti-TNFα purifiedwith either caprylic acid (▴) or sodium sulphate (▪). TNFα antisera (●)was used as a control.

FIG. 2 shows results of a direct ELISA for detection of anti-TNFα IgG inantisera (●) and IgG purified by way of caprylic acid precipitation (▴).

FIG. 3 shows results of an immunocytotoxicity assay (IOTA) testing theneutralising activity of Anti-TNFα Fragment (▪), Intact Anti-TNFα (▴)and TNFα antisera (●) using L929 cells. Starting concentration:Anti-TNFα Fragment—50 mg/ml; Intact Anti-TNFα—210 mg/ml; TNFα antiseraprotein concentration—86 mg/ml.

FIG. 4 shows the results of an ELISA comparing binding to murine TNFα byovine blood-derived intact PcAbs (anti-human TNFα IgG) and monoclonalantibody Infliximab. Anti-human TNFα IgG (●), Infliximab (▪), andnegative control (PCB) (▴).

EXAMPLES Example 1 Preparation of Ovine Antisera to Human TNFα

Mature human TNFα (hTNFα) (UniProtKB Accession No.: P01375) was obtainedfrom R&D Systems, Boehringer. The amino acid sequence is shown as SEQ IDNo. 1.

The immunogen for the primary immunisation of merino wether sheepcomprised Freund's complete adjuvant and 100 μg of hTNFα per sheep. Theprotein:adjuvant mixture was injected subcutaneously and equally into 6injection sites chosen for their proximity to the axillary, inguinal,and prescapular drainage lymph glands. Each sheep was reimmunized at28-day intervals with 100 μg of hTNFα and Freund's incomplete adjuvant,and blood samples were collected 14 days later at approximately 4 weeklyintervals at processing facilities at Turretfield Research Centre(Rosedale, South Australia, Australia) according to strict state andnational ethical guidelines for animal welfare. The animals were notterminally bled. A total of 10 mL of blood per kg of body weight can becollected from the external jugular vein without detriment to theanimal.

Ovine antisera was subsequently stored at −20° C.

Example 2 Purification of Polyclonal Ovine Antibodies to Human TNFα(Intact Anti-TNFα)

Two different methods for purification of ovine PcAb were used in orderto determine which method would co-isolate to a larger extent theefficient inhibitor of human trypsin, α1-anti-trypsin. Either caprylicacid precipitation, which precipitates albumin and keeps the IgG insolution, or the sodium sulphate precipitation that precipitates IgGwere used. The purified IgG was filtered and stored at −20° C. ready forinclusion in the proposed formulation for oral administration or forfurther characterisation.

The presence of protease inhibitors in antisera and the two IgGfractions purified using either caprylic acid or sodium sulphate wasassayed against trypsin and chymotrypsin by way of a colorimetric assay.The assay was based on the methods of Kakade M L et al. Determination oftrypsin inhibitor activity of soy products: a collaborative analysis ofan improved procedure. Cereal Chem 51: 376-381, 1974 (which isincorporated herein by reference) and measured cleavage ofNa-Benzoyl-DL-Arginine-p-Nitroanilide Solution (L-BAPNA) (colourless) top-nitroanaline (a yellow substrate) by trypsin.

Two-fold dilutions of inhibitor sample were diluted in 50 mMTris-buffer, pH 8.2, containing 20 mM CaCl₂ (100 μl final volume) acrossa 96-well plate (Grenier UV-Star). To each well, 100 μl of trypsin (0.2mg/ml diluted in 1 mM HCL) or chymotrypsin (1 mg/ml in WFI) solution wasadded followed by 100 μl of either L-BAPNA or NSLPN (2 mM in DMSO) fortrypsin or chymotrypsin, respectively. Reactions were terminated after 5minutes for the trypsin assay and after 60 minutes for the chymotrypsinassay with the addition of 50 μl stop solution (30% v/v acetic acid) andthe absorbance measured spectrophotometrically at 410 nm using OmegaPolarStar. Blank samples were prepared by adding the stop solution priorto the substrate solution. The average absorbance value was then plottedagainst the dilution, resulting in a dose response curve. Representativecurves are shown in FIG. 1, which demonstrate that the sodium sulphatepurification retains the α1-anti-trypsin inhibitor much better that thecaprylic acid precipitation procedure.

These two methods can also be used to assess the survival of proteaseinhibitors in the oral formulation after incubation in simulated gastricand intestinal fluids.

Example 3 Enzyme-Linked Immunosorbent Assay for Characterizing AntibodyBinding

The specific antibodies produced bound to multiple epitopes on thesurface of recombinant human TNFα (rhTNFα) but not to recombinant rodentTNFα. The avidity of binding was extremely high.

A direct ELISA assay was developed for detection of anti-TNFα IgG in theovine antisera and in the purified fraction of IgG (purified by way ofcaprylic acid precipitation) from this antisera (Intact Anti-TNFα).Immulon 4HBx microtiter plates were coated with 1 μg/mL hTNFα. Plateswere washed with 3 changes of phosphate-buffered saline (PBS) containing0.1% Tween 20 (PBST) and blocked for 1 hour at 37° C. with blockingbuffer (2.5% fetal calf serum diluted in PBS). Plates were washed andincubated for 1 hour at 37° C. with antisera at initial dilutions of1:1000, followed by 1:2 serial dilutions; washed with PBST; andincubated with a donkey anti-ovine IgG horseradish peroxidase conjugatefor 1 hour at 37° C. After further washing,3,3′,5,5′-Tetramethylbenzidine (TMB) liquid substrate solution wasadded, and the reaction was stopped after approximately 10 minutes bythe addition of 1.0 M HCL before reading the optical density at 450 nm.

The developed assay showed very low background which was tested usingpre-immune ovine serum (FIG. 2).

Example 4 Immunocytotoxicity Assay for Characterizing AntibodyNeutralization

The L929 mouse fibrosarcoma cell line (commercially available fromSigma-Aldrich, The Old Brickyard, New Road, Gillingham, Dorset, SP8 4XT,UK) was used to test the cytotoxic effects of TNFα as well as theneutralising ability of antibodies to TNFα. An assay was thereforedeveloped to test neutralisation of the cytotoxic effect of rhTNFα bythe ovine PcAb in the antisera, and by purified IgG from the antisera(Intact Anti-TNFα), and by fragments thereof (Anti-TNFα Fragment).

Anti-TNFα Fragment was prepared by subjecting a portion of the stock ofovine antisera of Example 1 to papain digestion. The Fab were present ata concentration of 10 g/L and about 10% of the total Fab were specificfor TNFα. An affinity chromatography step was not included in itsmanufacture. In the presence of excess of Fab, about 12 molecules of Fabbecome attached to each TNFα trimer.

As a challenging dose we used the 1090 hTNFα concentration of 13 ng/mldetermined from a cytotoxicity assay (data not shown). Briefly, L929cells containing twice the necessary challenging dose in DMEM wereco-incubated with an equal volume of various dilutions of hTNFα antiseraor Anti-TNFα Fragment or Intact Anti-TNFα for 24 h. As a positivecontrol (maximum killing), 2.5 μg/ml hTNFα was used. Antibody toxinneutralisation titres were estimated by colorimetric assays based oncell staining with neutral red.

Antibody toxin neutralisation titres were estimated by colorimetricassays based on cell staining with neutral red (representative curvesshown in FIG. 3).

The specific antibody concentration was calculated as follow:

Specific Ab conc [g/L]=[CCD(μg/L)−LC50(μg/L)]×[MW Av/(MWAg×BS)]×EC50×10⁻⁶

-   -   CCD (μg/L)—challenging dose=13 μg/L (LC90 determined from the        TNFα cytotoxicity on L929 cells)    -   LC50 (μg/L)=0.3 μg/L (determined from the TNFα cytotoxicity on        L929 cells)    -   BS—binding sites=2 for whole IgG    -   MW Ab=160 000 Da    -   MW Ag (TNFα)=51 000 Da

Taking the above into account, the specific PcAb concentration in theantisera was calculated at 2.9 g/L.

Example 5 Formulation of Intact Anti-TNFα

The choice of protease inhibitors to protect the PcAb of the invention(Intact Anti-TNFα) from digestion as well as the inhibitor's survival ingastric and intestinal fluids was assessed against trypsin andchymotrypsin using colourimetric assay, based on the method of Kakade ML et al. Determination of trypsin inhibitor activity of soy products: acollaborative analysis of an improved procedure. Cereal Chem 51:376-381, 1974. The trypsin assay is described above. The chymotrypsinassay utilised a colourless NSLPN substrate producing a yellow colour.

The two antacids in Table 1, magnesium hydroxide and aluminium hydroxidegel, were added to neutralise the low gastric pH, and thereby preventpepsin in the stomach from degrading the active components of IntactAnti-TNFα—the ovine IgG against TNFα and the egg white trypsin andchymotrypsin inhibitors. Other constituents were antimicrobialsmethylparaben and propylparaben which are believed to maintain a lowbioburden. The antifoaming agent simethicone was added to preventprotein denaturation when the suspension is mixed prior to use.Sweeteners in the form of sodium saccharin and mannitol reduce thebitterness of the two antacids. A flavouring agent, peppermint oil, wasused to improve the taste. A suspending agent, xanthan gum, was added tokeep all the above components in suspension.

It was experimentally determined that adding 200 mM Glycine to theformulation significantly improved stability of the suspension.

TABLE 1 Composition of Intact Anti-TNFα oral formulation. Concen-Category Material name tration Active IgG Intact Anti-TNFα   50 g/lcomponents Protease Egg white dried (EWD)   60 g/l inhibitor ExcipientsAntacid Magnesium hydroxide  23.4 g/l Dried aluminium  26.4 g/lhydroxide gel Antimicrobial Methylparaben (E218)    2 g/l Propylparaben(E216)  0.6 g/l Suspension Glycine (200 mM)   15 g/l stabilityAntifoaming Simethicone 16.89 g/l agent Sweeteners Sodium saccharin  0.4g/l Mannitol   21 g/l Flavour Peppermint oil  0.2 g/l Suspending Xanthangum    4 g/l agent

Example 6 Physicochemical Characterisation of the Composition Formulatedfor Oral Delivery

The physical appearance of the suspension and its purity were ensured bycarefully performing visual inspections. Sedimentation volume of thesuspension was determined by pouring 50 ml of the formulation into a 100ml measuring cylinder and the sedimentation volume was monitored andrecorded at different time intervals.

Duplicate results were obtained and the sedimentation volume wascalculated according to the equation:

F=Vu/Vo

Where F is sedimentation volume, Vu—ultimate height of sediment andVo—initial height of total suspension.

Within 48 hours of the beginning of the sedimentation experiment, nosedimentation was observed (Vu=0). Therefore, the pharmaceuticalsuspension remained very stable within 48 hours without any separation.Furthermore, after 48 hours the cylinder with the suspension was turnedupside-down and none of the layers were observed to be disposed at thebottom of the cylinder. This demonstrates that there were no layersforming within the suspension. In other word, there was no flocculationobserved within the prepared suspension during the 48-hour period of theexperiment.

Example 7 Case Study

A 27 year old white male on an emergency admission to hospital isdiagnosed as suffering from acute, severe ulcerative colitis which isconfirmed by colonoscopy and biopsy. The subject is immediatelyadministered an intravenous course of hydrocortisone but fails torespond over the next six days. There is concern that he might require atotal colectomy and, consequently, he is orally administered aformulation of the invention (e.g. as per Example 5). For the first twoweeks he receives 50 ml twice daily and, thereafter, 50 ml daily for afurther 12 weeks.

The patient makes a quick and excellent recovery based on his answers tothe UK bowel disease questionnaire (UK-IBDQ) and various clinicalparameters. In addition measured parameters such as C-reactive proteinreturn to normal values, and he experiences no serious adverse effects.At the end of the study there is significant mucosal healing as assessedby a second colonoscopy and biopsy.

Example 8 Synergistic Effects of the Compositions Formulated for OralDelivery

The following formulations are provided:

-   -   Formulation A: Intact Anti-TNFα;    -   Formulation B: Egg White Dried Protease Inhibitor;    -   Formulation C: Bowman-Birk Inhibitor (soybean);    -   Formulation D: Intact Anti-TNFα+Egg White Dried Protease        Inhibitor; and    -   Formulation E: Intact Anti-TNFα+Bowman-Birk Inhibitor (soybean).

25 patients with ulcerative colitis and 25 patients with Crohn's Diseaseprovide consent to be involved in a study to test the efficacy ofFormulations A-E. The patients are split into groups of 5 andadministered one of Formulations A-E (i.e. 5 patients with ulcerativecolitis are administered Formulation A and 5 patients with Crohn'sDisease are administered Formulation A, 5 patients with ulcerativecolitis are administered Formulation B and 5 patients with Crohn'sDisease are administered Formulation B, etc.). The dosage regime is 20ml (equivalent to 1 g of Intact Anti-TNFα) twice daily for 4 weeks, andonce daily thereafter.

Physicians determine that patients in both disease groups administeredFormulations D and E show a greater improvement and reduced symptomswhen compared with patients administered Formulations A−C. Colonoscopiesreveal an improvement in the surface layers of the intestinal tractpost-treatment. The improvement in patients administered Formulations Dand E is much greater than the improvement in patients administeredFormulations A−C, and much greater than the expected combinedimprovement of Formulations A+B and Formulations A+C (as determined byway of colonoscopy). Thus, the combination of PcAbs and proteaseinhibitors (e.g. EWD protease inhibitors and/or Bowman-Birk Inhibitors)yields unexpected synergistic effects.

Comparative Example 9 Comparative Analysis of Specific Antibody Titresfrom Sheep Serum (Ovine) & Hen Eggs

A study was undertaken to assess the concentration and avidities ofspecific IgY obtained from hen eggs in comparison with specific antibodyconcentrations from ovine antisera.

A group of 10 chickens and 5 sheep were immunised with humaninterleukin-6 (hIL-6, a pro-inflammatory cytokine like TNFα) and thetitres and avidities of the resultant specific PcAb was compared. Theaverage avidity constants were 1.3×10¹⁰ L/mol for chicken IgY vs3.1×10¹⁰ L/mol for the ovine antibodies. However, the levels of specificPcAb attained in the sheep (with an average titre of ≥1:200,000) weremore than ten times the titres found in egg yolk 1:20,000). This tenfoldor more difference in the concentration of specific PcAb was alsoapparent when sheep and hens were immunised with a number of otherimmunogens.

The above experiment shows the advantages of antibodies derived fromblood, and from an ovine source in particular.

Example 10 Comparative Analysis of Specific Antibody Titres Sourced fromSheep Serum (Ovine) & Cow's Milk (Bovine)

A study was undertaken to assess the potential of colostrum and milkfrom suitably immunised cows as a source of PcAb.

Cows were immunised with human TNFα and the titres of the resultingspecific PcAb determined first in the colostrum and then in serialsamples of milk. The maximum titre obtained in colostrum was 1:275,000(as compared with 1:800,000 in ovine antisera) and, after the firstmilking, levels rapidly fell to approximately 1:27,500.

Thus, blood-derived sources were shown to yield higher concentrations ofantibodies that bind to human TNFα when compared tomilk/colostrum-derived sources.

Example 11 Stability of Oral Antibody Formulations

The oral formulation of Example 5 was tested for antibody binding andneutralising activity following storage for approximately 12 months. Itwas shown that there was no deterioration in protease inhibitoractivity, and no change in the physical stability of the formulation,which retained antibody binding and neutralising activity.

Example 12 Efficacy of Antimicrobials of the Oral Antibody Formulations

The antimicrobial agents in the oral formulation of Example5—Methylparaben (E218) (concentration 2 g/L) and Propylparaben (E216)(0.6g/L)—were subjected to external antimicrobial testing to EuropeanPharmacopeia standards for such organisms as S. aureus, P. aeruginosa,E. coli, C. albicans and A. brasiliensis. Complete sterility was shown.

Example 13 Comparison of Blood-Derived Polyclonal Antibodies withMonoclonal Antibodies

A comparative antigen-binding assay was performed using blood-derived(ovine) polyclonal antibodies that bind to human TNFα and Infliximab(Schering-Plough Ltd), a monoclonal antibody that binds to human TNFα.

FIG. 4 shows that the monoclonal antibody, Infliximab, does not bind tomurine TNFα, whereas the ovine derived polyclonal antibodies raisedtowards human TNFα do.

The blood-derived polyclonal antibodies of the invention were shown tobind to and neutralise murine TNFα albeit at a concentrationapproximately 100-fold higher than that needed to neutralise human TNFα.No neutralisation of murine TNFα was observed by Infliximab, indicativeof an overall reduced neutralisation capability when compared toantibodies of the invention.

SEQUENCES SEQ ID No. 1VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQV YFGIIAL SEQ ID No. 2MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL SEQ ID No. 3MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCP SEQ ID No. 4VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVV PSEGLYLIYSQVLFKGQGCPSEQ ID No. 5 VRSSSRTP SEQ ID No. 6 HVVANPQAEGQLQWLNRR SEQ ID No. 7NGVELR SEQ ID No. 8 VPSEG SEQ ID No. 9 CPSTHVL SEQ ID No. 10 ISRIAVSYQTKSEQ ID No. 11 PCQRETPEGAEAK SEQ ID No. 12 DRLSAEINRPDYLDFASEQ ID No. 13 (Variant Human TNFα P84L)MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTLSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the present invention. Although the present invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biochemistry and biotechnology or related fields areintended to be within the scope of the following claims.

1. An antibody composition for oral administration comprising intactblood-derived polyclonal antibodies that bind to a human tumour necrosisfactor α (TNFα), and means for protecting the antibodies duringgastrointestinal transit.
 2. The antibody composition according to claim1, wherein the blood-derived polyclonal antibodies are ovineblood-derived polyclonal antibodies.
 3. The antibody compositionaccording to claim 1, wherein the blood derived polyclonal antibodiesare equine blood-derived polyclonal antibodies.
 4. The antibodycomposition according to any one of the preceding claims, wherein themeans for protecting the antibodies comprises at least a proteaseinhibitor.
 5. The antibody composition according to claim 4, wherein theprotease inhibitor is one or more selected from the Bowman-Birkinhibitor family of proteins.
 6. The antibody composition according toany one of the preceding claims formulated as a liquid.
 7. The antibodycomposition according to any one of the preceding claims, wherein themeans for protecting the antibodies comprises a means obtainable fromegg white.
 8. The antibody composition according to any one of thepreceding claims, wherein the means for protecting the antibodiescomprises egg white, preferably powdered egg white.
 9. The antibodycomposition according to any one of the preceding claims, wherein themeans for protecting the antibodies comprises one or more antacid(s).10. The antibody composition according to any one of the precedingclaims, wherein the means for protecting the antibodies comprises one ormore of: a. a polypeptide which binds specifically to and suppresses orinactivates the proteolytic activity of trypsin and/or chymotrypsin;and/or b. an antibody that binds to trypsin and/or chymotrypsin andsuppresses or inactivates the protease activity of said trypsin and/orchymotrypsin; and/or c. an antacid.
 11. The antibody compositionaccording to any one of the preceding claims, wherein the compositioncomprises an antacid and: a. a polypeptide which binds specifically toand suppresses or inactivates the proteolytic activity of trypsin and/orchymotrypsin; and/or b. an antibody that binds to trypsin and/orchymotrypsin and inactivates the protease activity of said trypsinand/or chymotrypsin.
 12. The antibody composition according to any oneof the preceding claims, wherein the means comprises one or more ofovomucoid, ovostatin, ovomacroglobulin, or combinations thereof.
 13. Theantibody composition according to any one of the preceding claims,wherein at least 5% of the total antibodies present in the compositionbind to TNFα.
 14. The antibody composition according to any one of thepreceding claims, wherein at least 10% of the total antibodies presentin the composition bind to TNFα.
 15. A method for manufacturing intactblood-derived polyclonal antibodies that bind to human tumour necrosisfactor α (TNFα), said method comprising obtaining a blood sample from anon-human mammal that has been administered an immunogen comprisinghuman TNFα or a fragment thereof.
 16. The method according to claim 15,wherein the non-human mammal is an ovine non-human mammal.
 17. Themethod according to claim 15 or 16 further comprising obtaining serumfrom the blood sample.
 18. The method according to claim 15, wherein thenon-human mammal is an equine non-human mammal.
 19. The method accordingto claim 15 or 18 further comprising obtaining plasma from the bloodsample.
 20. The method according to any one of claims 15-19 furthercomprising purifying antibodies using sodium sulphate precipitation orcaprylic acid precipitation.
 21. An antibody obtainable by the method ofany one of claims 15-20.
 22. An antibody composition according to anyone of claims 1-14 for use in treating an inflammatory disorder, whereinthe antibody composition is orally administered to a subject.
 23. Use ofan antibody composition according to any one of claims 1-14 in themanufacture of a medicament for treating an inflammatory disorder,wherein the antibody composition is orally administered to a subject.24. A method of treating an inflammatory disorder comprising orallyadministering an antibody composition according to any one of claims1-14.
 25. The antibody composition for use, use or method according toany one of claims 22-24, wherein the antibodies and the means forprotecting the antibodies are administered simultaneously.
 26. Theantibody composition for use, use or method according to any one ofclaims 22-24, wherein the antibodies and the means for protecting theantibodies are administered separately.
 27. The antibody composition foruse, use or method according to any one of claims 22-26, wherein theinflammatory disorder is septic shock.
 28. The antibody composition foruse, use or method according to any one of claims 22-27, wherein theinflammatory disorder is a gastrointestinal disorder, preferably anintestinal disorder.
 29. The antibody composition for use, use or methodaccording to any one of claims 22-28, wherein the inflammatory disorderis an inflammatory bowel disease, an intestinal infection, a graft vs.host disorder, a disorder caused by: non-steroidal anti-inflammatorydrugs, stress, alcohol, bowel surgery, ischaemia and reperfusion, foodallergy, or combinations thereof.
 30. The antibody composition for use,use or method according to any one of claims 22-29, wherein theinflammatory disorder is an inflammatory bowel disease selected fromulcerative colitis, Crohn's disease, or combinations thereof.
 31. Theantibody composition for use, use or method according to any one ofclaims 22-30, wherein the antibody composition is administered to asubject at a dose of 2 g or less daily.
 32. The antibody composition foruse, use or method according to any one of claims 22-31, wherein theantibody composition is administered to a subject at a dose of 1 g orless daily.
 33. A kit comprising an antibody composition according toany one of claims 1-14, and instructions for use of same.
 34. Afoodstuff comprising an antibody composition according to any one ofclaims 1-14.
 35. The foodstuff according to claim 34, wherein thefoodstuff is a dairy product, preferably a dairy product selected fromyogurt, cheese, or milk.
 36. The foodstuff according to claim 34 or 35further comprising a probiotic, and optionally a prebiotic.